Phytosphingosine alleviates acute gvhd by inducing th cell differentiation to Tr1 through GPR120/IL-10 axis

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for a broad range of hematologic diseases. Unfortunately, allogeneic HSCT is still associated with significant morbidity and mortality related to transplant complications, namely acute graft-versus-host disease (GvHD). Recent studies have uncovered a major role for microbial metabolites in the function of the host immune system by which they influence disease processes such as acute GVHD. Phytosphingosine (PHS) is a sphingomyelin substance and a sphingolipid that is known to be a major constituent of the plasma membrane of yeast; it is abundant in plants but also found in small amounts in the mammalian epidermis and small intestine. Previous studies have confirmed that PHS is effective in preventing moisture loss from the skin; regulates epidermal cell growth, differentiation, and apoptosis; and possesses bactericidal and anti-inflammatory activities, as well as the ability to regulate glucolipid metabolism. Whether PHS can alleviate acute GVHD and the associated mechanism is unclear. Here we showed that PHS is a novel and potent of donor T cells. To assess the differences in metabolome of acute GVHD, we performed LC−MS analysis using mice serum from control and acute GVHD mice. Principal component analysis revealed a distinct signature between the 2 groups, suggesting a significant biochemical changes. We further screened significantly different metabolites and displayed them in volcano plots. We found that PHS was significantly downregulated in the acute GVHD group and elevated in the control group, which was consistent with the heatmap of the hierarchic clustering analysis. In addition, the metabolic pathways of differentially expressed metabolites were analyzed, and bubble plot results revealed that sphingolipid metabolism was significantly different. Collectively, these results indicate that a low level of PHS in the recipient was correlated with the severity of acute GVHD. To determine whether PHS administration could alleviate severity acute GvHD, we first set up acute GVHD mice model to assess the effect of PHS by i.p. We found that the PHS significantly alleviated GVHD severity as reflected by reduced weight loss, clinical scores, and mortality. PHS administration was evident with lower serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST)， IFN-γ and TNF-αconcentrations compared with that of vehicle group. To understand the underlying mechanisms by which PHS reduces GVHD severity, we measured the activation, expansion, and differentiation of donor T cells in recipients' spleen, liver, small intestine and colon 3 weeks post-BMT. Administration of PHS significantly reduced the frequency and the number of CD4+ and CD8+ donor T cells in the spleen, liver and gut of recipients, suggesting reduced migration of donor T cells to target organs. Donor CD4+ T cells in the liver also produced less IFN-γafter treatment. In addition, administration of PHS significantly enhanced the frequency of Foxp3-IL-10+ Tr1 cells derived from donor CD4+ T cells in recipient spleen and liver. Taken together, administration of PHS ameliorated GVHD by reducing donor T cell expansion and Th1 differentiation in GVHD target organs while enhancing Tr1 cells generation or expansion. Phytosphingosine is a novel activator of GPR120. Deficiency of GPR120 in CD4+T cells resulted in more severe GVHD in mice. Treatment with PHS promoted CD4+T cell production of IL-10 by upregulating Blimp1 and enhancing glycolysis, which was regulated by mTOR, as same effect as GPR120 agonist, CpdA. Finally, we administrated PHS to recipient mice challenged by A20 cells, a lymphoma cell line originated from BALB/c mice. BALB/c mice that received A20 cells and a syngeneic transplant died within 21 days, and the inhibitor treatment had no impact on the survival of syngeneic transplant recipients. In contrast, mice that received A20 cells and an allogeneic transplant showed significantly better survival, and the inhibitor treatment further increased the survival of these mice. Our results indicated that PHS preserves the GVL effect in mice. Our findings demonstrate the role of PHS in regulating Th1 differentiation to Tr1 cells by GPR120/IL10 axis to inhibit acute GVHD development while preserving the GVL effect, which identifies PHS as a potential therapeutic target for treating acute GVHD.